Physical exercise is an important regulator of systemic glucose homeostasis in both healthy individuals and in people with insulin resistant diseases such as diabetes. This is because exercise increases the rate of glucose uptake into skeletal muscle, a major tissue responsible for peripheral glucose disposal. In addition to acute effects of exercise to increase glucose uptake into the contracting muscles, the period after exercise is characterized by an increase in the sensitivity of muscle glucose transport system to actions of insulin. These effects on muscle glucose uptake and insulin sensitivity are likely one of the most important health benefits of physical exercise. Despite the physiological importance of exercise in regulating glucose uptake in skeletal muscle, the molecular mechanisms leading to this phenomenon are not well understood. The overall goal of this project is to elucidate the mechanisms through which exercise increases glucose uptake in skeletal muscle, and increases the sensitivity of this process to insulin. It is well established that exercise effects on muscle glucose uptake involve the translocation of the GLUT4 glucose transporters from an intracellular site to the plasma membrane. Several critical experimental approaches will be integrated to investigate the molecular mechanisms that regulate exercise- stimulated GLUT 4 translocation and the post-exercise increase in muscle insulin sensitivity. First, the role of putative signaling molecules in contraction-stimulated glucose transport will be studied using an isolated muscle preparation. Second, muscle fractionation techniques will be used to generate plasma and intracellular microsomal membranes in order to investigate the regulation of GLUT4 vesicular trafficking with exercise. Third muscle-specific insulin receptor knockout mice and muscle-specific GLUT4 knockout mice will be studied to clearly define the function of these proteins in the regulation of glucose uptake and insulin sensitivity by exercise. These experiments should provide important information on the underlying molecular mechanism leading to the increases in skeletal muscle glucose uptake and insulin sensitivity that occur with exercise. Ultimately, these studies should provide us with a better understanding of glucose regulation during exercise in healthy people and in individuals with diabetes.